Inverse correlation between cyclic GMP and tyrosine aminotransferase degradation in rat hepatoma tissue culture cells

Vol. 105, No. 2, 1982 March 30, 1982

INVERSE

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

CORRELATION DEGRADATION Sarah

724-729

BETWEEN CYCLIC GMP AND TYROSINE AMINOTRANSFERASE IN RAT HEPATOMA TISSUE CULTURE CELLS

Taylor

Strinden'and

Robert

Stellwagen

H.

Department of Biochemistry University of Southern California School of Medicine Los Angeles, CA 90033

Several cyclic nucleotide phosphodiesterase inhibitors that increase tyrosine aminotransferase degradation in rat he atoma tissue culture (HTC) cellsincluding theophylline, caffeine, theobromine, ';- methyl-3-isobutylxanthine and papaverine-all lower cGMP levels in these cells while exerting variable effects on cAMP levels. In contrast, insulin slows tyrosine aminotransferase dearadation and increases cGMP levels when HTC cells are treated with the hormone for 4 hr in the absence of serum. The inverse relationship observed between cGMP and tyrosine aminotransferase degradation is consistent with a role for this cyclic nucleotide in the control of specific protein deqradation.

Several

compounds

frequently

diesterases-including lower

TAT'

(l-3).

although

they

previous

studies

HTC cells

is

do

correlate

with

shown

the

Other

(3).

on

degradation HTC cells

in

an

TAT

the

?Abbreviations culture.

of

phospho-

cGMP

shown cells

that

TAT,

terms

or

tyrosine

the of

cGMP

Obstetrics

dose

and

aminotransferase;

de-

and

and

have cGMP

the

Gynecoloqy,

HTC,

in

an

appears

response

when

Our

deqradation

CAMP

levels

to

TAT

(2).

change

CAMP

derivatives

of

TAT

of latter

Furthermore,

rate

synthesis on

the

increased

(3-5).

Mn zc

protein

papaverine-

degradation,

concentration Only

in

protein

theophylline

the

(3).

the

general

of in

and

general

effect

degradation

adding

used:

nucleotide

increasing

of

'Present address: Department of Wisconsin, Madison, Wisconsin 53705.

tissue

cyclic

affect

increase

have

hepatoma by

not

inhibition

level

studies

inhibit

specifically

do

that

by

increased

course

in

some

accompanied in

by

compounds

produce have

decrease

elevated

HTC cells

These

expected

TAT

in

to

1-methyl-3-isobutylxanthine,

theophylline,

activity

gradation

used

no levels

cultures,

University

hepatoma

unto time

influence are TAT

of

Vol.

105,

No.

degradation in

is

HTC

cells

not

in been

liver

(6) In

(9)

investigated the

inhibitors they

produce

have

found

levels

in

changes

in

cGMP

in

related

the

possibility

and

they be

EXPERIMENTAL .___

every to

of

also

show

explained

insulin

has

hepatocyte

the

a new that solely

cells

have

been

correlate

TAT

degradation.

some

drugs

cause

the

inhibition

in

to

slow

reported

and

to

TAT

degradation

general

protein

increase

cGMP

although

the

several

investigated

with

in

cGMP

inhibit

this

has

cells. insulin

for

to

(lO,ll),

of

role

by

ability

cultures

changes in

RESEARCH COMMUNICATIONS

shown

been

hepatoma

which

changes

its

effects

HTC

case,

been

to

in

study,

cGMP

that

inversely

and

also

compared

previously

present on

has

addition,

slices

AND BIOPHYSICAL

Insulin

(3).

(7,8).

In

cannot

slowed preferentially

degradation levels

BIOCHEMICAL

2, 1982

changes

levels

of

controlling changes of

to in cGMP,

These

results specific

in

cGMP

phosphodiesterase

levels

determine

TAT

whether We

degradation.

but

not further

protein in

CAMP,

are

support degradation,

HTC cells

which

phosphodiesterases.

PROCEDURES

HTC cells were grown in spinner or monolayer cultures as previously described (l-3). Logarithmically growing spinner cultures were usually pre-induced overnight with 1 LIM dexamethasome. In a typical experiment, a culture havinq 8 x lo5 cells per ml was divided into two or more spinner bottles and incubated for appropriate times in modified Swim's S-77 medium containing 10% bovine serum and appropriate test compounds. The cells were harvested by centrifugation for 2 min at 800 x g (4") and washed twice by resuspension in cold 0.14 M NaCl-0.01 M potassium phosphate buffer, pH 7.6. Monolayer cultures were maintained in plastic tissue culture dishes with inocula of l-2 x lo5 cells/cm' in a humidified incubator under 54 CO2 in air for at least 8 hr before addition of medium containing test substances. The cells were harvested after appropriate incubation times by scraping with a rubber policeman, after which the cells were separated from the medium by centrifugation and washed as described above. In experiments using insulin, HTC cells were collected by centrifugation; resuspended in serum-free medium supplemented with 0.1% bovine serum albumin, 1 LIM dexamethasone, 50 U/ml penicillin, and 50 ug/ml streptomycin; and maintained in spinner cultures from 18 hr prior to addition of 1 irM insulin throughout the experiment. Tyrosine aminotransferase degradation was measured as described earlier (3) either by precipitating radioactive pulse-labeled TAT from cell extracts followed by gel electrophoresis or by monitoring the loss of catalytically active TAT in the presence of 0.2 mM cycloheximide. These two methods yielded similar results (3). Cyclic nucleotide levels were determined as described earlier (3) in 5:: trichloroacetic acid-soluble fractions of cell lysates usinq the lZ51radioimmunoassay procedure reported by Steiner et al. (12) after chromatographic separation of CAMP and cGMP. Protein content and TAT enzymatic activity were measured as reported earlier (1). Theophylline, caffeine, theobromine, papaverine, bovine pancreatic insulin, bovine serum albumin, cycloheximide, and dexamethasone were from Sigma Chemical CO.; and l-methyl-3-isobutylxanthine was from Aldrich Chemical Co. Powdered tissue culture medium and penicillin/streptomycin were purchased from Grand Island Biological Co. Serum was obtained from KC Biologicals. 725

Vol. 105, No. 2, 1982 RESULTS

AND

and

to

ower

degradation

(l-3).

to

ine,

theophyl

changes

TAT

which this

to

decrease

example, with

under

the also

data

not

It nucleotide occur

an

inhibitory

0.1

support

not

in of

in with

surprising

effect by

soluble

and

tissues

(13 There can system rate

others

in

these

drugs,

also

inhibit

purified

particulate

guanylate

are

reports

activate

guanylate

1 mM sodium of

TAT

for

1 hr

no

significant

we

cyclase

(13).

I,

TAT

structure

with

at

all

these

CAMP

of

For treat-

the

either in

com-

vat-y.

while on

levels rate

and

generality

of

CAMP,

after

doses

phosphodiesterase

effect

the

level

1 or

of

5 hr.

HTC cells

TAT

degradation

1-3,

cGMP

and

TAT

evidence

degradation.

that

changes

in

degradation. are

commonly

levels

in

observed from

sodium

In cyclase

that

nitropruss

no

Carbachol 726

cGMP

effect

on at

to

nhibit

cyclic

We b e lieve the

case

from

the

HTC ce

elevate

used

HTC cells.

cyclase. guanylate

and

of

in

The

on

elevates

additional

cGMP

had

Table effects

between

cyclases

that

other

(Refs.

which

have

HTC cells

the

cGMP

Since

TAT

rate

degradation

(3).

levels

in

guanylate

nitroprusside

degradation

their

CAMP

soluble and

in

but

I provide

changes

(16,17);

several

data

correlation

TAT

in

circumstances

lower

on

the

I.

the similar

degradation

decreases

these

of

levels

examining

affect

increasino

pre-

(13).

by

has

Table

Table

that

they

by

in

inverse

results

tested

3 hr

rate

TAT

HTC cells;

result

an

in

shown

inhibitor

cGMP

mM papaverine

all

by

theophylline

lower

shown

for

drugs

under

because

reported

the

in

phosphodiesterases,

may

or

As

5 mM does

correlate

is

to

was

reported

the

do

pounds

the

increased

Furthermore, CAMP

of

by

inhibitors--

papaverineaere

HTC cells

the

increase

levels

at

conditions

the

the

mM papaverine

Theobromine all

with

with

0.04

However,

shown

levels

phosphodiesterase and

increase

produced

above.

cGMP

nucleotide

in

to

been

cGMP

treatment

CAMP.

our

on mentioned

pounds

RESEARCH COMMUNICATIONS

a phosphodiesterase

found

those

has

effect

activity

also

correlate

inhibitors

13),

cyclic

caffeine,

specific

was

identical

times

is

other

Theobromine,

Theophylline

ment

several

1 methyl-3-isobutylxanthine,

viously

of

AND BIOPHYSICAL

DISCUSSION

Theophylline including

BIOCHEMICAL

of

rat

theophylline,

liver

has

drug

can

1s

and

several

rat

de

and

related

com-

inhibit

(14,15,16). either

a concentration

this

crude

However, the

level of

been

of 10

in cGMP uM

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

Time

RESEARCH COMMUNICATIONS

(hr)

Gg&. The effects of insulin on cyclic nucleotide levels in HTC cells. The same cultures of HTC cells used for the experiment in Table II were used to determine the changes in CAMP (Part A) and cGMP (Part 6) in response to 1 pM insulin. Cyclic nucleotide levels from control (x-x) or insulin-treated (e-4) cells were determined as described previously (3). The results are plotted as the mean + standard error of duplicate determinations.

Table

I.

Experiment

Effects

of

Compound

various

phosphodiesterase

inhibitors

Added

Concentration

Number 1

2

3

(mM) None

nucleotide

Exposure

Time

(hr)

levels Cyclic

in AMP

HTC

cells

Cyclic

GMP

(fmol/mg)

(wol/mg) 1

0.86

+ 0.02

13.0

z 0.2

1

1

1.49

+ 0.03

11.7

2 1.5

Papaverine

0.1

1

1.04

+ 0.02

2.0

+ 1.0

Theophylline

1

1

1.22

f

4.7

+- 0.7

3

0.60

-+ 0.04

None

0.04

12.1

$ 1.2

l-Methyl-3-isobutylxanthine

1

3

1.70

-+ 0.08

9.1

+ 0.7

Papaverine

0.04

3

0.47

-+ 0.08

1.2

t

Caffeine

1

3

1.04

+ 0.02

6.4

+.~ 3.0

Theophylline

1

3

0.69

+- 0.01

4.0

f

1

0.09

-+ 0.003

1

0.10

+- 0.05

5

0.21

+- 0.01

48.2

+ 2.9

5

0.23

+ 0.02

30.7

T 0.1

None 5

None Theobromine

grown exposed averages 1 and

cyclic

l-Methyl-3-isobutylxanthine

Theobromine 4

on

______-

5

23.0 6.5

0.2

+- 3.5 + 2.0

Cyclic nucleotide levels were determined as described in MATERIALS AND METHODS for HTC cells in monolayer (Experiment 2) or spinner (Experiments 1, 3 and 4) cultures, The cells were to the indicated drug concentrations for the times shown. Results are presented as + standard errors of duplicate determinations derived from single cultures (Experiments 2) or two separate cultures (Experiments 3 and 4) for each condition.

727

1.0

Vol. 105, No. 2, 1982

BIOCHEMICAL

Table

II.

Effects

AND BIOPHYSICAL of

insulin

and

degradation Compound

Added

in

Concentration

RESEARCH COMMUNICATIONS

theophylline

on TAT

HTC cells (1lM)

None Insulin

7

Theophylline Insulin

1000 plus

TAT Half-life

(hr)

2.1

+

0.1

(8)

6.0

+

1.1

(8)

1.3

+

0.1

(4)

3.9

+ 0.6

(4)

1

theophylline

1000

Radioactivity in pulse-labeled TAT was determined by antibody precipitation and gel electrophoresis as previously described (3). Half-lives of degradation were calculated from the equation for a first-order process by using duplicate radioactivity measurements made at zero time and at 2 and 4 hr. The results are presented as the mean + standard error for the number of determinations given in parentheses.

elevates TAT

cGMP

in

levels

HTC cells

in

liver

(13).

We have

guanylate

cyclase

dependent

manner

in

HTC cells

slow

TAT

degradation

cGMP,

also

together

(19),

provide

cells

but

previously

elevates

strong

(9,18)

cGMP (3).

had

reported and

slows

All

evidence

for

of of

the

effect

that TAT

Derivatives (3).

no

on Mn'+,

either an

deqradation cyclic

a correlation

of

a dose-

such

above-mentioned

as

8-bromo-

results

between

cGMP

or

activator

in GMP,

cGMP

and

taken

TAT

de-

gradation. One a role --et

possible

in

al.

the (6)

known

to

that

ditions

for

cGMP However,

may

cGMP

insulin which

it

effect

does

levels

of

not the

theophylline

on

HTC cells involve rate

of

in

degradation

part,

clear

that

Insulin

hepatocyte cGMP

in

phenomenon has

been in

cultures HTC

cells

TAT effect

cGMP

cannot

effect

of

cGMP,

since

neither

protein

insulin

under

on

on for

theophylline,

con-

increase

in

degradation. of

protein

13).

(9).

same

actions

Mn?+, (l-3,

also

insulin

TAT

general

is slices

the

an

all

play

Spencer

liver

Furthermore,

insulin

may

Insulin

Thus,

account

it by

and 1)

II).

degradation

728

reported

(Figure

of

that

HTC cells.

degradation.

the

is

(10,ll)

(Table

the

general

this

preferentially

TAT

least

seems in

in

for

TAT.

degradation

slows

at

Even

on

increases it

also

function

insulin

TAT

cGMP

mediate,

changes

of

slow

to

the

(18,20). probably

effect

elevate

We find

reverses

physiological

insulin

degradation nor Although

8-bromothe

Vol. 105,

No.

actions

of

suggest

that

volve

insulin

cGMP.

gradation

are

BIOCHEMICAL

2, 1982

are

complex

the

preferential

The

mechanisms

currently

and

may

effect by being

AND BIOPHYSICAL

which

involve

of cGMP

a variety

the

hormone may

be

in

our

investigated

RESEARCH COMMUNICATIONS

on

of

mechanisms,

TAT

involved

in

our

data

degradation

could

in-

specific

protein

laboratory.

ACKNOWLEDGEMENTS __--.___ This Institute Sarah Science

work was (CA 12563), Taylor Strinden Laboratories

supported by research grants from the American Diabetes Association, was the recipient of predoctoral and the USC Cancer Center.

the

National and Sigma fellowships

Cancer Xi. from Bio-

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. il. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Stellwagen, R. H. (1974) Biochim. Biophys. Acta 338, 428-439. Stellwagen, R. H., Sailor, R. D., and Kohli, K. K. (1977) Biochem. Biophys. Res. Commun. 78, 1162-1167. Strinden, S. T. and Stellwagen, R. H. (1981) J. Cell. Physiol. 108, 231-237. Wicks, W. U., Barnett, C. A., and McKibbin, J. B. (1974) Fed. Proc. 33, 1105-1111. Stellwagen, R. H., Kohli, K. K., and Sailor, R. D. (1977) J. Cell. Physiol. 91, 51-61. Spencer, C. J., Heaton, J. H., Gelehrter, T. D., Richardson, K. I., and Garwin, J. L. (1978) J. Biol. Chem. 253, 7677-7682. Hershko, A., Mamont, P., Shields, R., and Tomkins, G. M. (1971) Nature New Biol. 232, 206-211. Gelehrter, T. D. and Emanuel, J. R. (1974) Endocrinology 94, 676-684. Illiano, G., Tell, G.P.E., Siegel, M. I., and Cuatrecasas, P. (1973) Proc. Nat. Acad. Sci., U.S.A. 70, 2443-2447. Spence, J. T., Merrill, M. J., and Pitot, H. C. (1981) 3. Biol. Chem. 256, 1598-1603. Earp, H. S. (1980) J. Biol. Chem. 255, 8979-8982. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972) J. Biol. Chem. 247, 1106-1113. Taylor, S. E. (1981) Ph. Il. Dissertation. The University of Southern California. Murad, F., Mittal, C. K., Arnold, W. P., Katsuki, S., and Kimura, H. (1978) Adv. Cyclic Nut. Res. 9, 145-158. Bijhme, E., Graf, H., and Schultz, G. (1978) Adv. Cyclic Nut. Res. 9, 131-142. Braughler, J. M., Mittal, C. K., and Murad, F. (1979) Proc. Nat. Acad. Sci., U.S.A. 76, 219-222. Tsai, S. C., Manganiello, V. C., and Vaughan, M. (1978) J. Biol. Chem. 253, 8452-8457. Pointer, R. H., Butcher, F. R., and Fain, J. N. (1976) J. Biol. Chem. 251, 2987-2992. Chrisman, T. D., Garbers, D. L., Parks, M. A., and Hardman, J. G. (1975) J. Biol. Chem. 250, 374-381. Czech, M. P. (1977) Ann. Rev. Biochem. 46, 359-384.

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de-